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Metagenomic Analysis of Biofilm in Dental Plaque
What are Biofilms? A biofilm can be defined as a group of microorganisms that stick together on a surface. These cells often produce a matrix called extracellular polymeric substance (EPS), which is also known as slime. Biofilm is mostly composed of DNA, protein, and polysaccharides. The stages of biofilm attachment are initial attachment, irreversible attachment, maturation I, maturation II, and dispersion. Biofilms are notoriously hard to get rid of and are responsible for many hard to manage infections in hospital settings. (3) What is Metagenomics? Metagenomics involve the simultaneous genetic analysis of all microorganisms collected in given environmental samples. It can produce a species diversity profile that allows analysis of microbial communities found in the natural environment. This method differs from traditional genomics because the latter relies on the sequencing of laboratory-cultivated cultures. Often many microorganisms cannot be cultivated in lab settings. Large-scale metagenomic studies could lead to therapeutic applications by increasing our understanding of the microbial ecosystems found in diseased areas. Overview/Introduction of Study Oral diseases often start with dental plaque formation, which results from accumulation of biofilm. This study describes the metagenome of the human oral cavity with a specific focus on supragingival dental plaque and cavities. Dental cavities are usually seen as holes in the teeth due to structural damage. Dental plaque is a sticky substance that adheres to the surface of teeth. It's usually comprised of bacteria, acid, food pieces, and saliva. Acids within the plaque cause damage to teeth enamel, causing tooth decay, which results in dental cavities or caries. The cavity is occupied by hundreds of bacterial species to maintain equilibrium in the oral ecosystem. Anaerobic gram-negative bacteria often inhabit subgingival plaque, which causes gingivitis and periodontitis. Supraginival dental plaque, formed by acidogenic and acidophilic bacteria, is responsible for dental cavities. (1) Metagenomic analysis was used in this study because it allows the study of entire microbe/bacterial communities within the supragingival dental plaques. It also allows a comparison between the biodiversity of samples of individuals with and without dental caries. Additionally, this study aimed to avoid biases resulting from PCR amplification and cloning techniques from previous studies by performing direct sequencing of oral metagenome DNA using 454 pyrosequencing. Methods and Materials Sampling For this study, supragingival dental plaques were gathered from 25 volunteers after informed consensus. These individuals were evaluated by dentists according to recommendations from the Oral health Surveys from the WHO. In volunteers with cavities, samples were obtained from non-cavity areas and individual cavities. The MasterPure complete DNA and RNA Purification Kit was used to extract the DNA. Eight samples from six individuals were used to pyrosequence based on similarities in age, oral hygiene, smoking habits, and mucosal health. They were divided into three groups: 1) healthy controls, which were two individuals that had never had any caries, 2) two individuals that have previous had caries and were treated, but had few caries at the time of sampling 3) two individuals that had many caries (8-15) and poor oral hygiene at sampling time. Six oral samples were taken from each individual. Additionally, samples from advanced and intermediate caries were collected, comprising of two additional samples. Sequence Analysis Artificially copied sequences and human sequences identified by MegaBlast in comparison to the human genome were removed from the initial data set. The remaining data were mapped using sequenced reference genomes with parameters by Nucmer and Promer v3.06 alignment algorithms. Taxonomic Assignment BLASTn allowed extraction of 16S rRNA from the metagenome data. Sequences less than 200 base pairs were taken out. A lowest common ancestor (LCA) algorithm was used to assign taxonomy to all open reading frames. Chen et. al’s 2010 oral microbiome database and NCBI RefSeq database of all bacterial and archaea genomes were also used for taxonomic assignment. Results Pyrosequencing Eight samples with various oral health statuses were sampled and direct pyrosequecing was performed. 1 Gbp of sequence was generated without any PCR or cloning. After human DNA was removed, the metagenome dataset contained 842 Mbp of sequence. An average of 129.5 Mbp of filtered high-quality sequences were obtained from each of six oral samples. An average of 32.5 Mbp of filtered high-quality sequences were obtained from the two cavity samples. Oral Metagenome Diversity This was approximated using three approaches: 1) Reads from the final dataset that matched 16S rRNA genes were assigned to various taxonomic levels. 4,254 16S rRNA sequences were obtained and about 73-120 genera were identified from the dental plaque samples. 2) LCA algorithm was used to classify taxonomic levels of reads from public databases. 1.5 million reads were assigned a taxonomic level. This confirmed results from the first approach, but suggests more diversity in taxonomic groups. 3) PhymmBL binding procedure was used to assign taxonomy to 1.94 million reads from the final dataset. The results supported the two approaches above. The overall results from all three methods showed the principle taxonomic groups without cloning or amplification of the sequences. Differences in the oral metagenome diversity in those with and without dental plaques were interesting. All three approaches showed that healthy individuals (those without dental plaques) tended to have high numbers of bacilli and gamma-proteobacteria. Diseased individuals (those with dental plaques) tend to have more anaerobic bacteria such as clostridiales and bacteroidetes. The two samples taken directly from cavities showed deficiency of bacilli and higher numbers of actinobacteria. Additionally, supporting earlier data, certain genera had higher tendencies to appear more in healthy individuals such as Rothio and Aggregatibacter (one of the most common species in individuals without dental plaques). Trichomonas tenax, a protozoan, was present in the oral cavity of volunteers with both poor oral hygiene and advanced periodontal disease. This particular microorganism seems to have a role in bronchopulmonary infections. Through an analysis of the abundance of related species in the metagenomic data, healthy and diseased individuals had high levels of certain bacteria in their oral cavities. In healthy individuals, close relatives of Aggregatibacter ''and ''Streptococcus sanguis were high, which agrees with previous research. In diseased individuals, Steptococcus gordonii ''and ''Leptotrichia buccalis were in high numbers. In all individuals, strains of Veillonella parvula appeared. Although large bacterial diversity exists in the oral metagenome, most bacteria fall under few taxonomic groups, and most are only present in low densities. Functional Significance of Diversity Application of the second and third approaches means that functionality of each bacterial group can be predicted. Bacilli contain many genes involved in defense mechanisms. Clostriales contain many cell motility genes. Bacilli were common in many healthy individuals while clostriales were common in diseased individuals. Samples from individuals with dental plaques tended to have clustered genomes taxonomically, which means similar functionality. Not all functionality of prevalent microorganisms are currently known. There are some similarities between the metagenomic diversity in the gut and oral with differences in prevalence of bacteria with certain functionalities. The relative numbers of microbacteria in the gut and oral cavities gave further insight into the ecosystem of these two linked ecosystems. Cavity Ecosystem Complexity The metagenomes of the cavity samples showed almost none Streptococcus mutans, which was previously believed to be the main cause of dental cavities. This is in agreement with other recent molecular studies. Since Veillonella has been found in all stages of cavity formation, synergism is likely. This bacteria in combination with S. mutans has been shown to cause more acid production than the two bacteria individually. Cavity development is considered to be a multimicrobial disease. Further research is needed to determine if different microbes are responsible for various stages of cavity development. This holds promise to develop vaccines against tooth decay. References 1. Belda-Ferre, Pedro, Luis David Alcaraz, Raúl Cabrera-Rubio, Héctor Romero, Aurea Simón-Soro, Miguel Pignatelli, and Alex Mira. "The Oral Metagenome in Health and Disease." The ISME Journal 6.1 (2011): 46-56. 30 June 2011. Web. 6 Dec. 2014. 2. Wikipedia article: Biofilm. Date accessed: Dec 6, 2014. 3. Wikipedia article: Metagenomics. Date accessed: Dec 6, 2014. 4. Nature web article: Focus on Metagenomics. Date accessed: Dec 6, 2014. 5. "Dental Cavities: MedlinePlus Medical Encyclopedia." U.S National Library of Medicine. National Institute of Health, 25 Feb. 2014. Web. 07 Dec. 2014.